Four novel ULBP splice variants are ligands for human NKG2D

Abstract

UL16-binding proteins [ULBPs, also termed as retinoic acid early transcripts (RAET1) molecules] are frequently expressed by malignant transformed cells and stimulate anti-tumor immune responses mediated by NKG2D-positive NK cells, CD8(+) alphabeta T cells and gammadelta T cells in vitro and in vivo. In this study, we identified four novel functional splice variants of ULBPs including ULBP4-I, ULBP4-II, ULBP4-III and RAET1G3 in HepG2 liver carcinoma cells, WISH human amnion cells, Hep-2 larynx carcinoma cells and K562 leukemia cells, respectively, by reverse transcription-PCR and T vector cloning strategy. Analysis of alignments of amino acid sequences of the splice variants illustrated that there were important modifications between splice variants and their individual parental ULBP. All ULBP4 splice variants (ULBP4-I, ULBP4-II and ULBP4-III) were type 1 membrane-spanning molecules and had the ability to bind with human NKG2D receptor in vitro. Ectopic expressions of ULBP4 and ULBP4 splice variants resulted in the enhanced cytotoxic sensitivity of target cells against NK cells, which could be blocked by anti-NKG2D mAb. Moreover, co-culture-free soluble forms of ULBP4 splice variants (their alpha1 + alpha2 ectodomains) and RAET1G3 (soluble splice variant of RAET1G2) with NK cells down-regulated the cell surface expression of NKG2D. Finally, immobilized in a plate-bound form of RAET1G3 stimulated NK cells to secrete IFN-gamma. Taken together, all the identified functional splice variants will help to advance our knowledge regarding the overall functions of ULBP gene family.

Anti-γδ TCR antibody-expanded γδ T cells: a better choice for the adoptive immunotherapy of lymphoid malignancies

“…Instead, they can recognize non-peptide antigens, such as phosphoantigens 18 and stress-induced proteins such as MICA/B, 19 ULBPs, 20 heat shock proteins and F1F0-ATPase, 21 via TCR and/ or non-TCR receptors. In vitro and in vivo studies have demonstrated that human cd T cells can kill various haematological and solid tumour cells expressing these antigens.…”

The NKG2D ligand ULBP4 binds to TCRγ9/δ2 and induces cytotoxicity to tumor cells through both TCRγδ and NKG2D

“…Stable transfectant EL4-ULBP4 was selected in RPMI-1640 supplemented (Gibco BRL) with 1 mg/mL G418. The transfected cells were stained with anti-ULBP4 mAb 8C9 previously generated in our laboratory, 27,28 followed by FITC-conjugated sheep anti-mouse IgG (Zhongshan) and sorted by flow cytometry. T-lymphoma cell line (J.RT3-T3.5), deficient in both TCR ␣ and ␤ chains (ATCC), was cotransfected with full-length human peripheral blood mononuclear cell (PBMC)-derived ␥9 and ␦2 chain (http://imgt.cines.fr/, 29 sequence shown in Table 1) (CDR3␦ region was OT3 nucleotide acid sequence).…”

“…In brief, a sequence encoding the extracellular domain of ULBP4 (aa 1-226) was amplified by polymerase chain reaction (PCR) using the primers U4f (5Ј-CGGAATTCATGCGAAGAATATCCCTGACTT-3Ј) and U4r (5Ј-GGCGGCCGCCTATTAGTGGTGGTGGTGGTGGTG-GTGGTGTCTAT CTGGTAGACTAGAAGAA-3Ј) from a previously cloned vector pET22b(ϩ)/ULBP4 26,27 and subcloned into the mammalian expression vector pEAK12, a kind gift from Dr Brian Seed (Massachusetts General Hospital and Harvard Medical School); the italicized portions of the sequences represent the restriction enzyme cutting site. Plasmid pEAK12/rULBP4 was stably transfected into HEK 293ET cells, and the fusion proteins were purified from culture supernatants using HisTrap kit (Amersham Pharmacia Biotech) following the instruction of the manufacturer.…”

Rhaphidophora korthalsii modulates peripheral blood natural killer cell proliferation, cytokine secretion and cytotoxicity

“…IFN-γ mediates NK cells’ cytolytic activity through the increase of the ULBP level of the tumors cells which then binds to the NKG2D [26]. Binding of RAET1G3 which is the variant of ULBPs expressed by HepG2 was found to stimulate NK cells to further enhance IFN-γ secretion [27]. This idea had raised the possibility that IFN-γ production stimulated by R. korthasii methanol extract might contribute to the activation of NK cells’ cytotoxicity against HepG2 and K562 [9-11].…”